The invention relates to oligosaccharides and libraries incorporaiting oligosaccharide. More particularly, the invention relates to oligosaccharides and libraries of oligosaccharides which employ amide and/or phosphodiester linkages for joining adjacent carbohydrate subunits.
Carbohydrates are known to mediate many cellular recognition processes. Carbohydrates can serve directly as binding molecules and, in such instances, are essential to the recognition process. A review of the biological role of carbohydrates with respect to cellular recognition phenomena is provided by Sharon et al. (Scientiic American, January 1993, 82). The emerging importance of glycobiology is further characterized by Mekelburger et al. (Angew. Chem. Int. Ed. Engl. 1992, 31, 1571) and by Dagani et al. (Chem. Eng. News, Feb. 1, 1993, 28).
Dysfunctional mediation of cellular recognition processes can lead to disease states. If a cellular recognition process is mediated by an oligosaccharide, then an absence or excess of such oligosaccharide can lead to a dysfunctional mediation of such process. The mediating oligosaccharide may be deficient or absent due to a deficiency of production or due to a high rate of catabolism. If rate of catabolism is excessive, then catabolically resistant analogs of the bioactive oligosaccharide may be preferred as drug candidates as compared to the native bioactive oligosaccharide.
Accordingly, what is needed is a library which includes analogs of known bioactive oligosaccharides. Such a library may be usefully employed for screening drug candidates.
Central requirements for the design of libraries of oligosaccharide analogs include the following:
(a) A need to maximize the potential of the designed oligosaccharides as ligand and drug candidates;
(b) A need to capitalize on existing highly sophisticates technology directed to the synthesis of oligopeptides and oligonucleotides in order to facilitate the rapid and efficient design and construction of oligosaccharides; and
(c) A need for flexibility with respect to synthesizing either single target molecules or large libraries of target molecules simultaneously.
Methodologies for synthesizing biopolymers are well developed for peptides, nucleic acids, and saccharides. Segments of oligopeptides and of oligonucleotides can now be routinely synthesized both in solution and in the solid phase, manually and/or on automated systems. The synthesis of such structures is facilitated by the availability of efficient techniques and sophisticated instrumentation for synthesizing peptide and phosphate bonds with high yields. The synthesis of oligopeptides and oligonucleotides is also facilitated by the absence of stereocenters in these linkages. In contrast, technology for the construction of oligosaccharides is comparatively less sophisticated and efficient. Synthetic methods for constructing oligosaccharides give comparatively lower yields and are complicated by the two isomer possibilities (xcex1 and xcex2) in glycoside bond formation.
Techniques and chemical methods for simultaneously synthesizing multiple oligopeptides, e.g. 100-150 completely different peptides having lengths of up to 20 amino acid residues, are reviewed by Jung, G. et al. (Angew. Chem, Int. Ed. Engl. 1992, 31, 367-383xe2x80x94incorporated therein by reference). Such techniques facilitate the construction of oligopeptide libraries.
Simon, et al. (Proc, Natl. Acad. Sci. USA, 1992, 89, 9367-9371) disclose oligopeptide analogs in which amino acid side chain groups are attached not to conventional peptide backbone carbons but to peptide backbone nitrogens. Such analogs are termed peptoids. Simon also discloses the construction of peptoid libraries as a modular approach to drug discovery. Simon""s oligopeptoids are shown by calculation to have greater conformational freedom as compared to conventional oligopeptides. Accordingly, oligopeptoids are thought to have greater potential as pharmaceutically useful binding ligands as compared to conventional oligopeptides having close sequence homology to such oligopeptoids.
Von Roedern et al. disclose a carbohydrate amino acid (Angew. Chem, Int. Ed. Engl. 1994, 31, 687-689). Although von Roedern discloses that carbohydrate amino acids may be coupled to peptides, he does not disclose that they may also be polymerized so as to form oligosaccharides.
A first aspect of the invention involves the molecular design and chemical synthesis of a class of carbohydrates designated as carbopeptoids (CPD""s). Glycopeptoids are preferred carbopeptoids. Carbopeptoids and glcopeptoids are oligosaccharides which employ peptide-like amide bonds for linking the various carbohydrate subunits within an oligomer assembly. Amide bond formation may be achieved by employing oligopeptide synthesis technology and instrumentation. The method allows for the design and synthesis of specific compounds for biological and pharmacological investigations. The method also allows for the generation of libraries of compounds for biological and pharmacological screening. Conventional screening techniques employed with respect to peptide and peptoid libraries (Simon et al., supra) may also be employed with respect to carbopeptoid libraries. The design takes advantage of the multifunctionality of carbohydrate subunits to maximize the binding properties of the molecules. The ease and high efficiency by which the peptide-like linkages can be constructed make the synthesis of these molecules a practical proposition. Furthermore, non-carbohydrate units may be inserted into the sequence making this approach even more flexible and versatile for the generation of new libraries of organic compounds.
More particularly, the invention is directed to a oligomeric carbopeptoid or glycopeptoid compound having carbohydrate amino acid subunits (CA""s) or glycoside amino acid subunits (GA""s) coupled to one another via an amide linkage. The amide linkage may be represented by the formula CA1-(CAxe2x80x94NH)-CA2. The amide linkage (COxe2x80x94NH) includes a carbonyl carbon and an amido nitrogen. A first carbohydrate amino acid subunit CA1 or glycoside amino acid subunit GA1 has an anomeric carbon bonded to the carbonyl carbon of the amide linkage. The anomeric carbon of the first carbohydrate amino acid subunit CA1 forms a C-glycosidic bond with the carbonyl carbon of the amide linkage and maintains the carbohydrate in a closed ring configuration. A second carbohydrate amino acid subunit CA2 has a non-anomezic carbon bonded to the amido nitrogen of the amide linkage. The second carbohydrate amino acid subunit CA2, like the first amino acid subunit CA1, may include an anomeric carbon bonded to the carbonyl carbon of a second amide linkage linking the second carbohydrate amino acid subunit CA2 to a third carbohydrate amino acid subunit CA3, etc. In this instance, the anomeric carbon of the second carbohydrate amino acid subunit CA2 forms a C-glycosidic bond with the carbonyl carbon of the amide linkage and maintains the carbohydrate in a closed ring a configuration. On the other hand, if the second carbohydrate amino acid subunit CA2 is a terminal subunit, then its anomeric carbon may form a hemiacetal, a hemiketal, or a glycoside.
The invention is also directed to a process for synthesizing the above oligomeric carbopeptoid or glycopeptoid compound. The synthetic process involves the coupling of two or more carbohydrate amino acid subunits (CA""s) or glycoside amino acid subunits (GA""s) to one another by means of amide linkages.
The invention is also directed to libraries of oligomeric carbopiaptoid or glycopeptoid compounds. Such libraries are employable for drug screening. Each oligomeric carbopeptoid or glydopeptoid compound includes at least two carbohydrate amino acid subunits (CA""s) or glycoside amino acid subunits (GA""s) coupled to one another via an amide linkage as indicated above. The invention is also directed to an improved process for synthesizing the above library of oligomers. The process employs an elongation step for coupling the subunits to one another to produce the oligomers. In the elongation step, two carbohydrate amino acid subunits (CA""s) or glycoside amino acid subunits (GA""s) are coupled to one another via an amide linkage as indicated above.
The invention is also directed to chemical intermediates for producing oligomeric carbopeptoids. A first chemical intermediate is a derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons. The anomeric carbon is substituted with a carboxyl radical. Each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, differentially protected amino, and hydrogen, with the proviso that at least one radical is a differentially protected amino. A second chemical intermediate is a derived carbohydrate amino acid similar to the first except that the non-anomeric carbons are substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, unprotected amino, and hydrogen, with the proviso that at least one radical is an unprotected amino and at least one radical is a blocked hydroxyl or amino.
A second aspect of the invention involves the molecular design and chemical synthesis of a class of carbohydrates designated as carbonucleotoids (CND""s). Carbonucleotoids are oligosaccharides which employ oligonucleotide-like phosphate bonds for linking the various carbohydrate subunits within an oligomer assembly. Phosphate bond formation may be achieved by employing technology and instrumentation developed for oligonucleotide synthesis. The phosphate bonds employed within carbonucleotoids are convenient linkages for coupling these units. The ease and high efficiency by which the oligonucleotide-like linkages can be constructed make the synthesis of these molecules a practical proposition.
The disclosed methods are characterized by their versatility and practicality. The methods may exploit conventional solid phase and automated synthesis techniques for producing carbopeptoids and carbonucleotoids in large scale.
More particularly, the second aspect of the invention is directed to an oligomeric carbonucleotoid molecule comprising carbohydrate C-glycoside subunits (CG""s) coupled to one another via a phosphodiester linkage. The phosphodiester linkage may be represented by the structure: CG1-C1xe2x80x2-(Oxe2x80x94PO(OH)xe2x80x94O)-CG2. The first carbohydrate C-glycoside subunit (CG1-C1xe2x80x2) has an anomeric carbon forming a C-glycosidic bond with a carbon C1xe2x80x2. In turn the carbon C1xe2x80x2 is bonded to the phosphodiester linkage. The second carbohydrate C-glycoside subunit CG2 has a non-anomeric carbon bonded to the phosphodiester linkage. The invention is also directed a process for synthesizing the oligomeric carbonucleotoid molecule. The process employs a coupling step wherein two or more carbohydrate C-glycoside subunits (CG""s) are coupled by means of a phosphodiester linkage as indicated above.
The second aspect of the invention is also directed to libraries of oligomeric carbonucleotoid molecules. The libraries are employable for drug screening. Each oligomeric carbonucleotoid molecule including at least two carbohydrate C-glycoside subunits (CG""s) coupled to one another by means of a phosphodiester linkage as indicated above. The invention is also directed to an improved process for synthesizing a library of oligomers. The process employs an elongation step wherein subunits are coupled to one another to produce the oligomers. The improvement is directed to the use of phosphodiester linkage linkages for linking the C-glycoside subunits as indicated above.
The second aspect of the invention is also directed to derived carbohydrate C-glycosides having an anomeric carbon and non-anomeric carbons. The anomeric carbon forms a C-glycosidic bond with carbon C1xe2x80x2. In turn, the carbon C1xe2x80x2 is bonded to an phosphoramidite. Each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, differentially protected hydroxyl, and hydrogen, with the proviso that at least one radical is a differentially protected hydroxyl. An alternative derived carbohydrate C-glycoside is similar to the above except that each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, unprotected hydroxyl, and hydrogen, with the proviso that at least one radical is an unprotected hydroxyl and at least one radical is a blocked hydroxyl. 